KR101021244B1 - Pharmaceutical composition comprising inhibitors of cell adhesion molecule isolated from Evodia fructus - Google Patents

Abstract

The invention ohsuyu (Evodia having the prevention and treatment of infectious diseases caused by ICAM-1 / LFA-1-mediated inflammatory diseases or rhinovirus cells or human immunodeficiency virus type -1 (HIV-1) caused by the adhesive fructus ) and a quinolone compound extracted from sewage oil . The present invention also relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases or infectious diseases using sewage oil extract or a quinolone compound derived therefrom as an active ingredient.

Fructus, intercellular adhesion factor-1 (ICAM-1), leukocyte function-related antigen-1 (LFA-1), cell adhesion, rhinovirus, human immunodeficiency virus type-1 (HIV-1), inflammation

Description

Pharmaceutical composition comprising inhibitors of cell adhesion molecule isolated from Evodia fructus}

The present invention provides a sewage extract having the effect of preventing and treating inflammatory diseases caused by ICAM-1 / LFA-1 mediated cell adhesion or infectious diseases caused by rhinovirus or human immunodeficiency virus type 1 (HIV-1), and It relates to a quinolone compound extracted from filthy milk. The present invention also relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases or infectious diseases using sewage oil extract or a quinolone compound derived therefrom as an active ingredient.

Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion receptor belonging to the Ig-superfamily of vascular endothelial cells, some lymphocytes and monocytes. are expressed at low density in monocytes (Springer, Nature, 346: 425-434, 1990; Rothlein et al., J. Immunol., 137: 1270-1274, 1986) IL-1, TNF-α or IFN-γ When treated with inflammatory cytokines or lipopolysaccharides (LPS), the expression of ICAM-1 is increased in multiple cell types (Hubbard and Rothlein, Free). Radic . Biol . Med ., 28: 1379-1386, 2000).

Leukocyte function associated antigen-1 (LFA-1; CD11a / CD18) is a ligand of ICAM-1 that is inactive in resting leukocytes but is mediated by T cell receptors or Activated by various stimulators such as phorbolester (Dustin and Springer, Nature , 341: 619-624, 1989; Rothlein and Springer, J. Exp . Med ., 163: 1132-1149, 1986).

On the other hand, in order for the white blood cells to infiltrate the inflammation site, the white blood cells must adhere to the vascular endothelial cells on the surface of the vascular endothelial cells. ICAM-1 is particularly expressed in vascular endothelial cells that have undergone inflammatory reactions, and promotes adhesion and penetration of blood cells such as monocytes, neutrophils, and lymphocytes to blood vessel walls. ICAM-1 also plays an important role in adhesion between platelets and vascular endothelial cells (Thomas and DeGraba, Neurology , 49: 15-19, 1997).

Cell adhesion is closely related to atherosclerosis, arthritis, osteoporosis, bacterial infections, cancer metastasis, and intractable chronic diseases such as angiogenesis, psoriasis, asthma, allergies, lupus or Crohn's disease (Poston et al., Am . J. Pathol ., 140: 665-673, 1992; Macchioni et al., J. Reumatol ., 21 (10): 1860-1864, 1994; Mason et al., Arthritis Rheum ., 36: 519-527 , 1993; Kling et al., Clin . Invest ., 71: 299-304, 1993). Among these, arteriosclerosis develops in the form of chronic inflammation when microscopic damage to the endothelial cells of the vessel wall is caused by hypertension, toxins or pathogens along with the mechanism of atherosclerosis due to excessive cholesterol concentration in the blood. In addition, monocytes in the blood penetrate into the blood vessel wall by cell adhesion factors expressed in endothelial cells, differentiate into macrophage from the blood vessel wall, and transform into foam cells that store cholesterol introduced into the blood. Progression of fibrosis leads to irreversible degeneration of narrowing blood vessels (Ross, Annu . Rev. Physiol., 57: 791-804, 1995; Christoper and Joseph, Cell , 104: 503-516, 2001). Therefore, cell adhesion factors play an important role in the process of inflammatory reaction and atherosclerosis, and various attempts have been made to develop inhibitors that block the expression of these adhesion factors between monocytes and endothelial cells.

These results suggest that ICAM-1 and LFA-1 mediated cell adhesion inhibitors are valuable as potential therapeutics for atherosclerosis and immune-related inflammatory diseases (Oppenheimer-marks and Lipsky, Clin . Immunol . Immunopathol ., 79: 203-210, 1996). In particular, the fact that ICAM-1 is important in the inflammatory response has been revealed through animal experiments with monoclonal antibodies of ICAM-1 and various disease models. Inhibition of binding between ICAM-1 and LFA-1 using monoclonal antibodies that directly correspond to ICAM-1 or LFA-1 has been shown to be effective in autoimmune diseases such as atherosclerosis, asthma, glomerulonephritis or arthritis In addition to antigen-specific signals through the T cell receptor, antigen-specific signals, such as ICAM-1 and LFA-1, are required for activation of T cells in the response (Cornejo et al., Adv . Pharmacol ., 39: 99-141, 1997; Patel et al., Circulation , 97: 75-78, 1998; Nishikawa et al., J. Exp . Med ., 177: 667-77, 1993; Kobayashi et al., Cell Immunol ., 164: 295-305, 1995; Nagase et al. Am . J. Respir . Crit . Care Med ., 152: 81-86, 1995).

 In addition, ICAM-1 plays an important role in viral infection. A cold virus, rhinovirus, has been demonstrated to infect using ICAM-1 as a receptor (Greve et al., Cell, 56: 839-847, 1989; Staunton et al., Cell, 56: 849-853 , 1989; Tomassini et al., Proc. Natl. Acad. Sci. (USA), 86: 4907-4911, 1989). In addition, human immunodeficiency virus type-1 (HIV-1) is also infected by the binding between ICAM-1 and LFA-1 and is known as a statin known as an inhibitor of binding of ICAM-1 and LFA-1. ) Compounds have been reported to inhibit the proliferation of HIV-1 virus (Giguere and Tremblay, J. Virology 78: 12062-12065, 2004). Therefore, the inhibitor of activity of ICAM-1 or LFA-1 can also be used as an antiviral agent of rhinovirus or HIV-1.

Currently, the development of ICAM-1 and LFA-1 mediated cell adhesion inhibitors has been targeted by their neutralizing antibodies, and only a few kinds of inhibitors have been reported from natural products. Abbott has reported ICAM-1 expression inhibitors as a synthetic product (Stewart et al., J. Med . Chem ., 44: 988-1002, 2001) . Antagonists of LFA-1 have been developed from Novartis and Roche (Welzenbach et al., J. Biol . Chem ., 277: 10590-10598, 2002, Gadek et al., Science , 295: 1086-1089, 2002), there are no reports of in vivo activity using disease animal models. On the other hand, from the natural products seco-limonids-based material ( Trichilia rubra , IC ₅₀ ; 1025 nM), cucurbitacins ( Concura scoparioides , IC ₅₀ ; 0.181.36 mM) and adzanthromycin (adxanthromycins, Streptomyces sp. Na-148, IC ₅₀ ; 1.56.5 mM), and the like. It has been reported as a substance that inhibits LFA-1 mediated cell adhesion (Musza et al., Tetrahedron, 50: 11369-11378, 1994; Musza et al., J. Net . Prod ., 57: 1498-1502, 1994 Nakano et al., J. Antibiotics , 53: 12-18, 2000).

On the other hand, Evodiae Fructus is a dried, immature fruit belonging to the Rutaceae, which has a spicy and bitter taste (Namba T., Colored Illustration of Wakan-Yaku, Hoikusha Publishing, Osaka, 1980, I, 233-234 ). Since ancient times, filthy milk has been used for strong stomach, analgesia, indigestion, abdominal pain, earth and sand, constipation and gusts, colic, musculoskeletal pain, uterine contraction, convergence and sterilization, and some baths. In addition, various types of essential oils, fatty oils, bitter taste limonoids and non-flavored indole alkaloids, quinolone alkaloids, beta carboline alkaloids, and the like were separated from the sewage oil, It is known to increase cerebral blood flow, uterine contraction, vasodilation, angiotensin II receptor antagonism and inhibitory effect on the growth of Helicobacter pylori (Wen-Fei C., J. Nat. Prod., 1996, 59, 374- 378; King CL, J. Nat. Prod., 1980, 43, 577-582 ;, Lee HS, Phytother.Res. 1998, 12, 212-214; Rho, TC, Bio. Pharm. Bull., 1999, 22 , 1141-1143).

As such, various pharmacological activities have been reported for sewage, but there are no reports and studies on ICAM-1 / LFA-1 mediated cell adhesion inhibitory activity.

The present inventors have searched for ICAM-1 and LFA-1 mediated cell adhesion inhibitors from natural resources, and as a result, sewage extract, fractions or quinolone compounds derived therefrom inhibit ICAM-1 and LFA-1 mediated cell adhesion. The present invention has been completed by elucidating that there is an effect.

One object of the present invention is ICAM-1 / LFA-1 mediated, obtained by extracting sewage oil ( Evodia fructus ) with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms and mixed solvents thereof. It is to provide a sewage extract for the prevention and treatment of inflammatory diseases caused by cell adhesion or infectious diseases caused by rhinovirus or human immunodeficiency virus type 1 (HIV-1).

Another object of the present invention is an inflammatory disease or linovirus or HIV-1 caused by ICAM-1 / LFA-1 mediated cell adhesion comprising the sewage extract or the compound of Formula 1 or 2 as an active ingredient. It is to provide a pharmaceutical composition for the prevention and treatment of infectious diseases.

In order to achieve the above object, in one embodiment, the present invention provides ICAM-, which is obtained by extracting sewage oil ( Evodia fructus ) with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms and mixed solvents thereof. The present invention relates to a sewage extract for the prophylaxis and treatment of inflammatory diseases caused by 1 / LFA-1 mediated cell adhesion or infectious diseases caused by rhinovirus or human immunodeficiency virus type-1 (HIV-1).

Method for obtaining a sewage extract in the present invention is as follows. The sewage oil pulverization is prepared from a lower alcohol having 1 (C ₁ ) to 4 (C ₄ ) carbon, such as water, methanol, ethanol and butanol, in volumes of about 2 to 20 times, preferably about 3 to 5 times, dry weight. A polar solvent or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10 thereof is used as the elution solvent, and the extraction temperature is 20 to 100 ° C, preferably at room temperature, and the extraction period is about 12 hours to 4 days, Preferably, extraction is performed using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction for three days. Preferably, the extract is filtered 1 to 5 times by cold extraction and subjected to filtration under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C., preferably at room temperature using a vacuum rotary condenser, and soluble in water, lower alcohols, or a mixed solvent thereof. One crude sewage extract can be obtained.

The sesame oil extract of the present invention inhibited the adhesion between sICAM-1 and THP-1 cells (expressed by LFA-1), which is a human monocyte cell line (LFA-1 expression), at a concentration of 50 μg / ml as shown in the following example (FIG. 1). . Therefore, it is suitable for the prevention and treatment of inflammatory diseases or infectious diseases.

The sewage oil extract of the present invention is extracted from the sewage oil with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof, and then selected from the group consisting of hexane, chloroform and ethyl acetate. Fractionation may be further included using an eluting solvent. Preferably, the present invention relates to a chloroform fraction obtained by repeatedly extracting the sewage oil extract sequentially with hexane and chloroform as a solvent.

In a further step of the present invention, the crude sewage crude extract is suspended in distilled water, and then, by adding a nonpolar solvent such as hexane, ethyl acetate or chloroform in a volume of about 1 to 100 times, preferably about 1 to 5 times the suspension, 1 It can be obtained by extracting and separating the nonpolar solvent soluble layer from times to 10 times, preferably from 2 times to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis , 3rd Ed. p6-7, 1998).

Specifically, after the crude sewage oil extract is suspended in water, an equivalent amount of ethyl acetate can be obtained by continuous extraction of each sewage oil solvent by continuous extraction, and more specifically, after the crude sewage oil extract is suspended in water, Equivalent amounts of ethyl acetate can be added to obtain ethyl acetate soluble fraction and water soluble fraction.

The ethyl acetate fraction of sewage oil of the present invention inhibited the adhesion between sICAM-1 and THP-1 cells of human monocyte cell line by 70% or more at a concentration of 50 μg / ml as shown in the following example (FIG. 1). Therefore, it is suitable for the prevention and treatment of inflammatory diseases or infectious diseases.

Inflammatory diseases in the present invention are systemic lupus erythematosus, scleroderma, ulcerative colitis, Crohn's disease, atopic dermatitis, psoriasis, anaphylaxis, dermatitis, diabetic retinopathy, retinitis, macular degeneration, uveitis, conjunctivitis, stroke, coronary artery disease , Arteriosclerosis, myocardial infarction, unstable angina, vasculitis, vascular stenosis, kawasaki disease, giant cell arteritis, arthritis, arteriosclerosis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergy, diabetes, diabetic nephropathy, nephritis, nephritis Disorders selected from the group consisting of Sjogren's syndrome, Crohn's disease, autoimmune pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometritis, rhinitis, tumor metastasis, transplant rejection and chronic prostatitis However, it is not limited thereto (Marlin, et al., Cell 51: 813-819, 1987; Dustin, et al., J. Immunol . 137: 245-254, 1986; Dustin, et al., Immunol . Today 9: 213-215, 1988; Poston et al., Am . J. Pathol . 140 : p665-673, 1992; Macchioni et al., J. Reumatol. 21 (10) : p 1860-1864, 1994; Mason et al., Arthritis Rheum . 36 : p519-527, 1993; Kling et al., Clin . Invest . 71 : p 299-304, 1993; US Patent No. 5,629,162; Korean Patent Application No. 10-2007-7006370).

In addition, the infectious disease in the present invention includes an infectious disease caused by rhinovirus or HIV-1.

In another embodiment, the present invention is directed to an inflammatory disease or rhinovirus or HIV-1 caused by ICAM-1 / LFA-1 mediated cell adhesion comprising the sewage extract or the compound of Formula 1 or 2 as an active ingredient. It relates to a pharmaceutical composition for the prevention and treatment of infectious diseases.

[Formula 1]

[Formula 2]

Quinolone compounds of Formulas 1 and 2 may be used in the form of a pharmaceutically acceptable salt, and include all salts, hydrates, and solvates prepared by conventional methods. Extraction, separation and purification of the quinolone compounds from filthy oil according to the present invention are as follows.

The ethyl acetate soluble fraction is separated by silica gel column chromatography using a mixed solvent of dichloromethane and methanol. At this time, it is preferable to use a solvent of dichloromethane and methanol 100: 1 to 1: 1 (v / v). ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity of the isolated active fractions was measured, and the fractions with the highest inhibitory activity were collected and separated by silica gel column chromatography using a mixed solvent of n-hexane and ethyl acetate. do. At this time, n-hexane and ethyl acetate are preferably used a solvent of 5: 1 to 1: 6 (v / v). The highest inhibitory fractions were collected and separated by reverse phase column chromatography using 70%, 80%, 90% and 100% acetonitrile as eluent. From the final fractions obtained, the fractions with high ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity were collected, and the compounds of Formulas 1 and 2 were separated by HPLC (high performance liquid chromatography) using 85% methanol and acetonitrile. Can be.

As in the following Examples, the IC ₅₀ value of the adhesion inhibitory activity of sICAM-1 and THP-1 cells of the compound of Formula 1 was measured to 43 ㎍ / ㎖, the IC ₅₀ value of the compound of Formula 2 was 47 Μg / ml. In addition, the compound of the present invention effectively inhibited adhesion between sICAM-1 and THP-1 cells, a human monocyte cell line, as the concentration increased. As a positive control, lovastatin was used, and lovastatin inhibited more than 60% at a concentration of 10 ㎍ / ml (Fig. 2). Accordingly, the compounds of formula 1 or 2 of the present invention are suitable for the prevention and treatment of inflammatory diseases or infectious diseases.

Fructus extract, Fructus fraction, or the compounds of Formulas 1 and 2 of the present invention are characterized in that ICAM-1 and LFA-1 mediated cell adhesion inhibiting activity of blood cells such as monocytes, neutrophils, and lymphocytes adhere to and invade vascular endothelial cells. By inhibiting it can be usefully used as a medicine for the prevention and treatment of inflammatory diseases. In addition, ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity can be useful as a drug for the prevention and treatment of viral infections.

The quinolone compound of the present invention was used by extraction, separation and purification from sewage weed, but can be obtained by all conventional methods, and commercially available reagents can be used. The sewage oil extract of the present invention or the compound of Formula 1 or 2 is derived from sewage oil, which is a drug that has been used for a long time, so there is no problem such as toxicity and side effects.

Pharmaceutical compositions for the prevention and treatment of inflammatory diseases or infectious diseases of the present invention is 0.0001 to 10% by weight, preferably 0.001 to 10% by weight of the sewage extract, sewage fraction, or the compound of formula 1 or 2 with respect to the total weight of the composition It is included in 1% by weight.

The sewage oil extract of the present invention, the sewage oil fraction, or a composition comprising the compound of Formula 1 or 2 may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The compositions according to the invention can be used in the form of oral formulations, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, respectively, according to conventional methods. have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose in the extract or fraction. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, glidants such as magnesium stearate or talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances or preservatives, in addition to water and liquid paraffin, which are commonly used as simple suspensions, solvents, emulsions or syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations or suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter or glycerogelatin may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Fructus extract, Fructus fraction, or the compound of Formula 1 or 2 of the present invention inhibits ICAM-1 and LFA-1 mediated cell adhesion and prevents the adhesion and penetration of blood cells such as monocytes, neutrophils and lymphocytes to vascular endothelial cells. Since it is effective in reducing, it can be usefully used as a medicament for the prevention and treatment of inflammatory diseases and infectious diseases infected using ICAM-1.

The present invention is explained in more detail based on the following examples, but the present invention is not limited thereto.

Example  One. From sewage  Extraction, Separation and Purification of Quinolone Compounds

1-1. From sewage  extraction

Sewage (seed) was washed with water, dried in the shade, and then powdered by Waring brand. To 1.2 kg of powdered sewage oil, three times methanol was added to the dried sewage oil weight, and extracted at room temperature for 7 days and then filtered. The filtrate was concentrated under reduced pressure to obtain a crude extract.

In order to isolate and purify the active substance from the crude extract, the crude sewage extract was suspended in water, and then the same amount of ethyl acetate was mixed and fractionated to obtain an ethyl acetate soluble fraction and a water soluble fraction. The obtained fractions were measured for cell adhesion inhibitory activity by the method of Example 2 below, showing the best intercellular adhesion inhibitory activity in the ethyl acetate layer.

1-2. Of ethyl acetate layer  Separation and Purification

The ethyl acetate layer obtained in Example 1-1 was concentrated under reduced pressure (54.7 g), and silica gel was prepared using a step gradient solvent system composed of dichloromethane: methanol = (100: 0 to 1: 1). The active fraction was separated using column chromatography.

The fraction having the highest intercellular adhesion inhibitory activity in the active fraction was adsorbed onto SiO ₂ , and then n-hexane: ethyl acetate = (5/1 to 1/6, v / v) was used in a gradient concentration solvent system. Secondly, the active fractions were separated by column chromatography on silica gel.

The fraction having the highest intercellular adhesion inhibitory activity among the active fractions was 70%, 80%, 90% and 100% acetonitrile as the elution solvent, and the active fraction was subjected to reversed-phase column chromatography (ODS gel). Was separated. 90% acetonitrile eluted fraction having the highest inhibitory activity in the fraction was flowed at 5 ml / min using 85% methanol, and high performance liquid chromatography (HPLC, YMC J'sphere ODS H-80, 150 × 20 mm, 4 mm) to obtain the active substances 1 (5 mg) and 2 (32 mg), which elute at 34 and 42 minutes.

1-3. Structural Analysis of Active Substances

Compound 1, which was completely isolated and purified, was a colorless powder. The UV spectrum showed absorption bands of 200, 215, 238, 321, and 333. The EI-MS spectrum showed molecular ion peaks at m / z 313, ion peaks by McLafferty rearrangement were observed at m / z 173, and quinolone alkaloids such as m / z 186 and m / z 144. Characteristic fragments of were observed. ^{In the 1} H-NMR spectrum, N-methyl groups at δ 3.74, conjugated olepic protons of δ 6.24 (1H, s, H-3), and δ 7.37-7.66 (3H, m, 6-, 7-, 8- Signals due to quinolone skeletons such as aromatic protons of H) and δ 8.45 (1H, dd, J = 6, 2.1 Hz, 5-H) were observed. Δ 2.72 (2H, t, J = 7.5 Hz, H-1 ′), δ 1.69 (2H, m, H-2 ′), δ 1.43 (2H, m, H-3 ′), δ 1.26 (14H , m, H-4'-10 ') and δ 0.88 (3H, t, J = 7.1 Hz, H-11') signal due to the side chain (signal) was observed. ^{In the 13} C-NMR spectrum, 21 carbon signals were observed, and compound 1 was 1-methyl-2-undecyl-4 (1H) -quinolone (1-methyl-2-undecyl- 4 (1H) -quinolone) (Sugimoto T, Miyase T, Kuroyanagi M, Ueno A. Limonoids and quinolone alkaloids from Evodia rutaecarpa Bentham. Chem . Pharm. Bull . 1988 , 36 (11), 4453-4461.).

Completely purified compound Compound 2 is a colorless oil which shows the same UV spectrum as compound 1. In the EI-MS spectrum, the molecular ion peak was shown at m / z 339, and the ion peak by McLafferty rearrangement of m / z 173 and fragments such as m / z 186 and m / z 144, as in compound 1, were observed. It was observed that it was a quinolone alkaloid compound. ^{In the 1} H-NMR spectrum, an olefinic proton of δ 5.34 (2H, m, H-8 ', -9') was observed, indicating that there was one double bond in the side chain, and δ 2.01 (4H). , m, H-7 ', -10') methylene proton signals were observed. In addition, 23 carbon signals were observed in the ¹³ C-NMR spectrum, and compound 2 was identified as evocarpine by comparing the above instrumental analysis data with literature values (Sugimoto T, Miyase T, Kuroyanagi). M, Ueno A. Limonoids and quinolone alkaloids from Evodia rutaecarpa Bentham. Chem . Pharm . Bull . 1988 , 36 (11), 4453-4461.).

Example  2. sICAM -1 and THP Inhibitory Activity Between -1 Cells

Recombinant sICAM-1 (R & D Systems, Abingdon, UK) was diluted to 10 [mu] g / ml (in PBS), and then 100 [mu] l was added to a 96-well plate and reacted at 4 [deg.] C. for 12 hours. After washing once with PBS, 100 μl of 10 mg / ml BSA was added and blocked for 1 to 2 hours at room temperature. THP-1 cells (1 × 10 ⁷ cells / ml) were reacted at 37 ° C. for 30 to 60 minutes after BCECF-AM (5 μM) was added. After THP-1 cells were washed with PBS and treated with LFA-1 / 2 antibody (500 ng / ml) for 10-20 minutes at 37 ° C, THP-1 cells and samples were coated with recombinant sICAM-1. 100 μl of each well of the plate was added and reacted at 37 ° C. for 30 to 60 minutes. After the reaction, unbound THP-1 cells were removed and washed three times with PBS. Cells were solubilized with PBS (1% Triton X-100) and fluorescence (ex. 485, em. 592) was measured.

Methanol extract and ethyl acetate fraction of sewage oil inhibited the adhesion between sICAM-1 and THP-1 cells by more than 35 and 70%, respectively, at a concentration of 50 μg / ml (FIG. 1).

IC ₅₀ values of the quinolone compounds represented by Formulas 1 and 2 of the present invention were 43 μg / ml and 47 μg / ml, respectively. Compounds of the present invention effectively inhibited adhesion of sICAM-1 and THP-1 cells with increasing concentration (FIG. 2).

Example  3. Formulation

3-1. Powder  Produce

Sewage oil extract and its fractions or 0.1 g of evocarpine, 1.5 g of lactose and 0.5 g of talc were mixed and filled into an airtight cloth to prepare a powder.

3-2. Manufacture of tablets

A tablet was prepared by direct tableting method after mixing sewage oil extract and its fraction or 0.1 g of evocarpine, 7.9 g of lactose, 1.5 g of crystalline cellulose and 0.5 g of magnesium stearate. The total amount of each tablet is 500 mg, of which the active ingredient content is 50 mg.

3-3. Manufacture of powder

A powder is prepared by mixing well the sewage extract and its fraction or 0.1 g of evocarpine, 5 g of corn starch and 4.9 g of carboxy cellulose. 500 mg of powder was put into the hard capsule to prepare a capsule.

4-4. Injection preparation

Sewage oil extract and its fractions or 0.1 g of evocarpine, a suitable amount of sterile distilled water for injection, and a pH adjusting agent were prepared in the above-described content of ingredients per ampoule (2 ml) according to a conventional method for preparing injections.

4-5. Liquid  Produce

A sewage oil extract and its fractions or 0.1 g of evocarpine, 10 g of isomerized sugar, 5 g of mannitol and an appropriate amount of purified water were dissolved in each of the ingredients in purified water according to a conventional method for preparing a liquid, and then a suitable amount of lemon flavor was added. After mixing the mixture was added purified water to adjust the whole to 100 ㎖ by adding purified water, and then filled in a brown bottle and sterilized to prepare a liquid.

Figure 1 shows the degree of inhibitory activity of methanol extract (MeOH) and ethyl acetate fraction (EtOAc) of sewage oil inhibit the adhesion between soluble ICAM-1 (sICAM-1) and THP-1 cells.

Figure 2 shows the degree of inhibitory activity of the compounds of formula 1 and 2 isolated from the ethyl acetate fraction of sewage oil inhibit the adhesion between soluble ICAM-1 (sICAM-1) and THP-1 cells.

Claims (8)

A sewage oil extract containing the compound of Formula 1 or 2 as an active ingredient, obtained by extracting sewage oil ( Evodia fructus ) with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof. Pharmaceutical composition for the prevention and treatment of infectious diseases caused by rhinovirus or human immunodeficiency virus type-1 (HIV-1) comprising. [Formula 1]

[Formula 2]

The composition of claim 1, wherein the lower alcohol solvent is selected from the group consisting of methanol, ethanol and butanol. The method of claim 1, wherein the sewage oil extract is extracted from a sewage oil ( Evodia fructus ) with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms and a mixed solvent thereof, and then composed of hexane, chloroform and ethyl acetate. A composition obtained by further comprising fractionating with an eluting solvent selected from the group consisting of: The composition of any one of claims 1 to 3, further comprising a pharmaceutically acceptable carrier. Pharmaceutical composition for the prevention and treatment of infectious diseases caused by rhinovirus or HIV-1 comprising a compound of formula (1) or (2) as an active ingredient. [Formula 1]

[Formula 2]

6. The composition of claim 5 further comprising a pharmaceutically acceptable carrier. The composition of claim 5, wherein the compound of Formula 1 or Formula 2 is obtained by extractive separation from sewage oil. delete

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